Aflatoxin B1 is a potently carcinogenic fungal metabolite which frequently contaminates peanuts,corn, and other agricultural products. The health hazard is of worldwide concern. We seek to identify the crucial DNA lesion responsible for the genotoxicity of aflatoxin B1. It is metabolized to the 8,9 epoxide, a synthesis of which has been developed in this laboratory. The epoxide reacts with DNA with extraordinary efficiency to give a thermally unstable cationic adduct at the N7 position of deoxyguanosine. The cationic adduct has a short half- life in vivo apparently because it is substrate for enzymatic repair processes. However, a minor fraction of the cationic adduct undergoes base-catalyzed cleavage of the guanine to give a formamidopyrimidine (FAPY) adduct. The FAPY adduct is highly persistent in vivo and therefore is generally believed to be more important lesion. This project will test the hypothesis that the cationic adduct, although intercalated, is recognized by repair proteins because it causes significant DNA bending whereas the FAPY adduct escapes detection by repair proteins. The test will involve structural studies of nucleoside, oligonucleotides and high DNA polymers bearing cationic and FAPY lesions including assessment of bending at the site of the lesion. FAPY adducts are conformationally and configurationally complex. Probably the most significant of these isomerization processes involves atropisomers termed FAPY I and II. The structures of FAPY I and II will be deduced including assignment of absolute configurations. A sensitive assay for FAPY structures will be developed which will be used to assay FAPY presence in genomic DNA and to distinguish FAPY I from FAPY II. Three collaborations represent important components of this proposal. One with Dr. Dinshaw J. Patel (Sloan Kettering) will oligonucleotide. For this project adducted oligonucleotide will be synthesized with 15N and/or 13C isotopic substitution at key locations. A second is with Dr. John M. Essigmann (MIT); this will involve studies of replication and repair of site-specific cationic and FAPY lesions. The final collaboration is with Dr. John D. Groopman (Johns Hopkins) and will use the FAPY assay to probe FAPY structures in the DNA of animal and human populations exposed to aflatoxin. The possibility will be examined that one of the FAPY forms and/or certain sequence contexts may be unusually resistant to repair. Overall, the proposed project on DNA adducts of aflatoxin B1 will provide a structural basis for understanding the biological activity of this potent carcinogen.